CN107727621B

CN107727621B - Method for detecting miRNA (micro ribonucleic acid) by using enzyme-labeled DNA (deoxyribonucleic acid) logic system

Info

Publication number: CN107727621B
Application number: CN201710910583.5A
Authority: CN
Inventors: 刘海云; 祝琳; 岳森; 何涛; 于京华
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2017-09-29
Filing date: 2017-09-29
Publication date: 2020-04-17
Anticipated expiration: 2037-09-29
Also published as: CN107727621A

Abstract

The patent discloses a method for detecting miRNA by an enzyme-labeled DNA logic system, and relates to the field of fluorescence detection of miRNA. By designing complementary probes, the specific recognition of the target is achieved without adding corresponding labels and specific enzymes. The characterization of optical signal amplification is realized by utilizing the fluorescence enhancement characteristic after protoporphyrin and G-quadruplex are combined, and the aim of detecting a target object is fulfilled. The NOT gate output is achieved by disrupting the binding using complementary, spontaneously-occurring strand displacement reaction probes. The method has low detection cost and high sensitivity.

Description

Method for detecting miRNA (micro ribonucleic acid) by using enzyme-labeled DNA (deoxyribonucleic acid) logic system

Technical Field

mirnas are widely present in various animals and plants, and participate in a variety of important life activities. By 6 months 2014, Release 21.0, the latest miRNA database of Sanger institute, has included 28645 miRNA precursors of 223 species, 35828 mature mirnas. At present, more than two thousand mirnas have been found in humans, accounting for 1% of the human genome, and exert regulatory effects on the expression of 30% of genes. Its regulation and control range is not limited to normal physiological process in human body (such as cell proliferation, differentiation, development and apoptosis, etc.), and it also has close relationship with heart disease and tumor occurrence and development, etc.

Background

Abnormal expression of miRNA leads to disease generation and physiological abnormality, and in many cancer cells such as breast cancer cells and colorectal cancer cells, the expression level of miRNA changes, possibly serving as a cancer suppressor gene and a protooncogene. In addition, abnormal expression of mirnas has also been detected in many other diseased cells or tissues. Therefore, timely detection of miRNA in tissue or cell samples is helpful for people to further understand the relationship between miRNA and disease development, and a new idea is provided for early diagnosis of diseases.

At present, common methods for detecting mirnas include a Northern Blotting method, a microarray method, a fluorescent labeling method, a quantitative-reverse transcription (PCR) method, a rolling circle amplification method, a gene chip technology, and the like. These methods have many problems after being improved, such as complicated process, expensive instrument, high experiment cost, and poor sensitivity, and thus have some limitations in clinical application. The methods all show inevitable defects, so that a method is needed to be developed, different specimens and even miRNA with low abundance can be sensitively and quantitatively detected, high homologous miRNA can be respectively homologized, the operation is convenient, and expensive equipment or reagents are not needed. This experiment attempted to develop a simpler and more efficient method for establishing a DNA logic system that could be free of enzyme labeling, which did not require a long analysis time and could observe the results by fluorescence signal intensity changes.

Disclosure of Invention

The invention aims to solve the technical problem of constructing a miRNA detection method of an optical signal DNA logic system without enzyme markers by using a DNA strand displacement technology;

a method for detecting miRNA by an enzyme-labeled DNA logic system is characterized by comprising the following steps:

(1) preparation of the principal solution

Tris-EDTA-KCl buffer: dissolving 0.0605 g of tris (hydroxymethyl) aminomethane, 0.0146g of ethylenediamine tetraacetic acid, 5.84 g of sodium chloride and 0.375 g of potassium chloride in sterile water, fixing the volume to a 50 mL volumetric flask, storing at 4 ℃ for later use, and adjusting the pH to 8.0 when in use;

6 mM protoporphyrin solution: dissolving 0.1879 g protoporphyrin in sterile water, diluting to a constant volume in a 50 mL volumetric flask, storing at 4 ℃ in a dark place for later use, and performing the whole process under the dark condition;

10% ammonium persulfate: 0.2 g of ammonium persulfate is taken and added with 2 mL of sterilized water, and the mixture is uniformly mixed and stored at the temperature of 4 ℃ for standby.

(2) Probe design

The corresponding G1, G2 and I chains were designed according to the target.

(3) Construction of DNA logic System

The target miRNA, G1, and G2 used were centrifuged separately in the rnase-free environment. And (3) performing low-temperature operation, adding 15 mu L of TEK buffer solution, 1 mu M G1, G2 and 10 mu L of target miRNA into a centrifuge tube, mixing uniformly, and centrifuging.

(4) Fluorescence detection

And (3) carrying out fluorescence intensity signal test, wherein the excitation wavelength is fixed to be 410 nm, the emission wavelength is 550-750 nm, the width of the excitation slit is 20 nm, and the width of the emission slit is 20 nm.

The above solution was heated to 88 ℃ for reaction for 10 minutes, slowly cooled to room temperature, and 5. mu.L of 6 mM protoporphyrin solution was added thereto and reacted at room temperature for 1 hour to conduct YES gate fluorescence intensity signal test. 10. mu.L of 4. mu. M I strand was added to the tube, and reacted at room temperature for 1 hour to conduct a NOT gate fluorescence intensity signal test.

The invention has the advantages of

(1) Establishing an enzyme-label-free DNA logic system according to the strand displacement reaction and the fluorescence characteristic of protoporphyrin without carrying out the previous labeling step;

(2) the miRNA is detected by using a fluorescence technology, so that the sensitivity is high, the experiment steps are convenient and fast, and the result can be obtained quickly;

(3) the method has the advantages of low detection cost, high sensitivity and good specificity.

Drawings

FIG. 1 is an experimental schematic of the YES gate of the method described herein.

FIG. 2 is an experimental schematic of the NOT gate of the method described herein.

FIG. 3 is a probe design for the methods described herein.

Detailed Description

For a better understanding of the invention, the following further illustrates the invention with reference to examples and drawings, but the invention is not limited to the following embodiments.

Example 1

(1) preparation of the principal solution

Tris-EDTA-KCl buffer: 0.0605 g of tris (hydroxymethyl) aminomethane, 0.0146g of ethylenediamine tetraacetic acid, 5.84 g of sodium chloride and 0.375 g of potassium chloride are taken, dissolved in sterilized water, and are added into a volumetric flask with a constant volume of 50 mL, and the volumetric flask is stored at the temperature of 4 ℃ for standby application, and the pH value is adjusted to 8.0 when the volumetric flask is used.

6 mM protoporphyrin solution: taking 0.1879 g protoporphyrin, dissolving with sterilized water, diluting to a constant volume in a 50 mL volumetric flask, storing at 4 ℃ in a dark place for later use, and performing the whole process under the dark condition.

(2) Probe design

The corresponding G1, G2 and I chains were designed according to the target.

(3) Construction of DNA logic System

(4) Fluorescence detection

Example 2

The detection procedure was the same as in example 1, except that: the initial concentrations of the I solutions were 4. mu.M, 2. mu.M, 0.4. mu.M, 0.3. mu.M, 0.2. mu.M, 0.1. mu.M and 0.05. mu.M, respectively.

Sequence listing

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Claims

1. a method for detecting miRNA without enzyme-labeled DNA logic system is characterized in that comprising the following steps:

1.1 Preparation of the main solution Tris-EDTA-KCl buffer: take 0.0605 g of tris, 0.0146 g of ethylenediaminetetraacetic acid, 5.84 g of sodium chloride, 0.375 g of potassium chloride, dissolve in sterile water, 6 mM protoporphyrin solution: take 0.1879 g of protoporphyrin, dissolve in sterilized water, and dilute to a 50 mL volumetric flask. Store in a dark environment at 4 ºC for later use, and carry out the whole process under dark conditions;

1.2 Probe Design

According to the target sequence, the corresponding G1, G2, and I chains are designed. G1 and G2 are rich in G bases to form the G-quadruplex part, and the target and I chain have necessary parts, which play the role of initiating the strand displacement reaction. These two parts are fixed in the system, and other sequences in the chain are designed accordingly according to the different target miRNAs;

1.3 Building a DNA logic system

In an RNase-free environment, centrifuge the target miRNA, G1 and G2 used separately, operate at low temperature, add 15 µL Tris-EDTA-KCl buffer to the centrifuge tube, 1 µM G1, G2 and 10 µL each of the target miRNA , mix, centrifuge;

1.4 Fluorescence detection

Fluorescence intensity signal test was performed, the excitation wavelength was fixed at 410 nm, the emission wavelength was 550-750 nm, the excitation slit width was 20 nm, and the emission slit width was 20 nm;

The above solution was heated to 88 ºC, reacted for 10 minutes, slowly lowered to room temperature, added 5 µL of 6 mM protoporphyrin solution, reacted at room temperature for 1 hour, and tested for YES gate fluorescence intensity. The reaction was carried out at room temperature for 1 hour, and the fluorescence intensity signal test of NOT gate was performed.

2. the method for detecting miRNA according to a kind of enzyme-free DNA logic system of claim 1, is characterized in that, utilizes the characteristic that the fluorescence intensity will obviously rise after protoporphyrin is combined with G-quadruplex, and takes the optical signal as the final result. The output establishes the DNA logic system.

3. the method for detecting miRNA according to a kind of non-enzyme-labeled DNA logic system according to claim 1, is characterized in that, through necessary part control to initiate strand displacement reaction, destroy the G-quadruplex structure constructed before, make fluorescence intensity greatly decline.